Abstract: Usher syndrome (USH) is the most common form of inherited deaf-blindness, with a prevalence of 1/6.000. Inherited as an autosomal recessive trait, it affects about 15,000 people in the United States and is responsible for 6% of early childhood deafness. Usher syndrome is classified under three clinical subtypes (USH-1, -2 and -3) according to the severity of the symptoms. Approximately 2/3 of the patients with USH suffer from USH2 and USH1, of whom 75% have mutations in the USH2A, USH1D and 1F genes. Because USH affects both major senses, it is a severely debilitating condition, and intense research is crucial to improve coping strategies and develop therapies for the patients. It is particularly devastating during the current pandemic, with social distancing and the wearing of masks making communication nearly impossible. Treatment for USH is limited to cochlear implants, and there is no treatment for the blindness. Development of an effective therapeutic approach for USH has been challenging due to the large size of USH genes. Therefore, there is an unmet need to develop alternative therapeutic strategies. The goal of this project is to develop and test novel therapy approaches for treating recessive deafness in human hair cells of inner ear organoids derived from human induced pluripotent stem cells (hiPSCs) and in USH mouse models by establishing genome editing-based therapeutic strategies for USH and to lay the foundation for moving genome editing approaches closer clinical trials. We have chosen to focus on the most common mutations in the three major USH genes with the following reasons: 1) The USH2A, 1D, and 1F genes are the most common and important USH genes which are responsible for more than 70% human USH cases with significant clinical application; 2) Due to their large size, traditional gene augmentation or addition therapy is hampered as its coding sequence far exceeds the packaging capacity of standard gene therapy vectors; 3) All three USH2A, 1D, and 1F genes contain similar multiple repetitive domains with in frame common mutations in their protein structures, making them potential targets for exon-skipping-based therapies (see Preliminary data); 4) We have obtained exciting data demonstrating restoration of hearing in an Ush2a mouse model using exon skipping strategy with an available mouse model of USH2A and have successfully generated hiPSCs from Usher patients carrying the most common mutations of USH, and established optimized protocols for generation of large numbers of human inner ear organoids with the production of human hair cells derived from these hiPSCs. This proposal leverages the exceptional deafness genomics information and genome editing expertise of collaborators for the development of novel treatments for HL. In this proposal, we will build on our accomplishments and preliminary data by proposing to complete the following specific aims: 1) to develop CRISPR/Cas9- based exon-skipping strategies to res...